This invention relates to compounds which are inhibitors of the interaction between the integrin xcex14xcex21, also known as Very Late Antigen-4 (VLA-4) or CD49d/CD29, and its protein ligands, for example Vascular Cell Adhesion Molecule-1 (VCAM-1) and fibronectin. This invention further relates to processes for preparing such compounds, to pharmaceutical compositions containing them and to their use in methods of therapeutic application.
xcex14xcex21 is a member of the integrin family of heterodimeric cell surface receptors that are composed of noncovalently associated glycoprotein subunits (xcex1 and xcex2) and are involved in cell adhesion to other cells or to extracellular matrix. There are at least 14 different human integrin xcex1 subunits and at least 8 different xcex2 subunits and each xcex2 subunit can form a heterodimer with one or more xcex1 subunits. Integrins can be subdivided based on their xcex2 subunit composition. xcex14xcex21 is one of several xcex21 integrins, also known as Very Late Antigens (VLA).
The interactions between integrins and their protein ligands are fundamental for maintaining cell function, for example by tethering cells at a particular location, facilitating cell migration, or providing survival signals to cells from their environment. Ligands recognised by integrins include extracellular matrix proteins, such as collagen and fibronectin; plasma proteins, such as fibrinogen; and cell surface molecules, such as transmembrane proteins of the immunoglobulin superfamily and cell-bound complement. The specificity of the interaction between integrin and ligand is governed by the xcex1 and xcex2 subunit composition.
Integrin xcex14xcex21 is expressed on numerous hematopoietic cells and established cell lines, including hematopoietic precursors, peripheral and cytotoxic T lymphocytes, B lymphocytes, monocytes, thymocytes and eosinophils [Hemler, M. E. et al (1987), J. Biol. Chem., 262, 11478-11485; Bochner, B. S. et al (1991), J. Exp. Med., 173, 1553-1556]. Unlike other xcex21 integrins that bind only to cell-extracellular matrix proteins, xcex14xcex21 binds to VCAM-1, an immunoglobulin superfamily member expressed on the cell surface, for example on vascular endothelial cells, and to fibronectin containing the alternatively spliced type III connecting segment (CS-1 fibronectin) [Elices, M. J. et al (1990), Cell, 60, 577-584; Wayner, E. A. et al (1989). J. Cell Biol., 109, 1321-1330].
The activation and extravasation of blood leukocytes plays a major role in the development and progression of inflammatory diseases. Cell adhesion to the vascular endothelium is required before cells migrate from the blood into inflamed tissue and is mediated by specific interactions between cell adhesion molecules on the surface of vascular endothelial cells and circulating leukocytes [Sharar, S. R. et al (1995). Springer Semin. Immunopathol., 16, 359-378]. xcex14xcex21 is believed to have an important role in the recruitment of lymphocytes, monocytes and eosinophils during inflammation. xcex14xcex21/ligand binding has also been implicated in T-cell proliferation, B-cell localisation to germinal centres, haemopoeitic progenitor cell localisation in the bone marrow, placental development, muscle development and tumour cell metastasis.
The affinity of xcex14xcex21 for its ligands is normally low but chemokines expressed by inflamed vascular endothelium act via receptors on the leukocyte surface to upregulate xcex14xcex21 function [Weber, C. et al (1996), J. Cell Biol., 134, 1063-1073]. VCAM-1 expression is upregulated on endothelial cells in vitro by inflammatory cytokines [Osbom, L. et al (1989) Cell, 59, 1203-1211] and in human inflammatory diseases such as rheumatoid arthritis [Morales-Ducret, J. et al (1992). J. Immunol., 149, 1424-1431], multiple sclerosis [Cannella, B. et al., (1995). Ann. Neurol., 37, 424-435], allergic asthma [Fukuda, T. et al (1996), Am. J. Respir. Cell Mol. Biol., 14, 84-94] and atherosclerosis [O""Brien, K. D. et al (1993). J. Clin. Invest., 92, 945-951].
Monoclonal antibodies directed against the xcex14 integrin subunit have been shown to be effective in a number of animal models of human inflammatory diseases including multiple sclerosis, rheumatoid arthritis, allergic asthma, contact dermatitis, transplant rejection, insulin-dependent diabetes, inflammatory bowel disease, and glomerulonephritis.
Integrins recognise short peptide motifs in their ligands. The minimal xcex14xcex21 binding epitope in CS-1 is the tripeptide leucine-aspartic acid-valine (Leu-Asp-Val) [Komoriya, A., et al (1991). J. Biol. Chem., 266, 15075-15079] while VCAM-1 contains the similar sequence isoleucine-aspartic acid-serine [Clements, J. M., et al (1994). J. Cell Sci., 107, 2127-2135]. The 25-amino acid fibronectin fragment, CS-1 peptide, which contains the Leu Asp-Val motif, is a competitive inhibitor of xcex14xcex21 binding to VCAM-1 [Makarem, R., et al (1994). J. Biol. Chem., 269, 4005-4011]. Small molecule xcex14xcex21 inhibitors based on the Leu-Asp-Val sequence in CS-1 have been described, for example the linear molecule phenylacetic acid-Leu-Asp-Phe-D-Pro-amide [Molossi, S. et al (1995). J. Clin. Invest., 95, 2601-2610] and the disulphide cyclic peptide Cys-Trp-Leu-Asp-Val-Cys [Vanderslice, P., et al (1997). J. Immunol., 158, 1710-1718].
More recently, in WO 96/00581, Publi. date Jan. 11, 1996, and WO96/20216, Publi. date Jul. 4, 1996, cyclic peptides containing the Leu-Asp-Val sequence have been reported to inhibit the binding of xcex14xcex21 integrin to VCAM-1 or fibronectin.
A few small non-peptidic Leu-Asp-Val surrogate compounds have been reported in WO 94/02445, Publ. date Feb. 3, 1994 to inhibit xcex14xcex21-induced adhesion.
More recently, non-peptidic compounds of formula I which can be orally adminstered, and which inhibit VCAM/VLA4 binding have been reported in PCT application WO96/22966. 
The preferred compounds are those in which in formula I, R1xe2x80x2 is an urea derivative, R2xe2x80x2 is hydrogen, R3xe2x80x2 is an alkyl or substituted alkyl, R4xe2x80x2 is dimethoxyl phenyl or benzo dioxol-5-yl and Y is CO.
There remains a continuing need for alternative compounds which inhibit the interaction between VCAM-1 and fibronectin with integrin VLA-4 and, in particular, for compounds which can be adminstered by an oral route.
We have now found a group of compounds containing a substituted phenoxy group which inhibit this interaction.
According to one aspect of the present invention there is provided a compound of formula (II) 
wherein:
R1 is in the para or meta position and is 
where R2 and R3 are each independently selected from hydrogen, nitro, C1-6 alkyl, C3-6 cycloalkyl, C2-6alkenyl, C2-6alkynyl, C1-4 alkoxy, C1-6 alkylamino, C1-6dialkylamino, C1-6alkylC1-4alkoxyl, C1-6alkylaminoC1-6alkyl, amino, cyano, halogeno, trifluoromethyl, xe2x80x94CO2R12, and xe2x80x94CONR12R13, where R12 and R13 are independently selected from hydrogen or C1-6 alkyl, or R2 and R3 together with the phenyl to which they are attached form a 9 or 10 membered bicyclic ring system;
R4 is C1-4alkyl;
R5 is selected from hydrogen and C1-4alkyl;
R6 is selected from C1-6 alkyl, C1-4alkyl(C4-6)cycloalkyl, C1-6alkyl(C1-6)alkoxyl, C1-6alkylS(C1-6)alkyl, C1-4alkylsulphonyl(C1-4)alkyl, 
where q is an integer from 1 to 6 and R14 is halogeno;
R7 is selected from C1-6alkyl, C1-8alkoxylcarbonyl, C2-6 alkenyl, 1,3-benzodioxol-5-yl and aryl optionally substituted by one or more substituents selected from C1-4 alkoxy, C1-6 alkyl, cyano, halogeno, and trifluoromethyl;
R8 is aryl, heteroaryl, a bicyclic heteroaryl ring system linked to the nitrogen via a ring carbon or a 9 or 10 membered bicyclic ring system linked to the nitrogen via a ring carbon and each ring is optionally substituted with up to two substituents, which may be the same or different, and are selected from C1-6 alkyl, C1-4 alkoxy, C1-4alkylthio, C1-6alkylC1-4alkoxyl, C1-6alkylaminoC1-6alkyl, hydroxy, xe2x80x94CO2H, (CH2)pOH, where p is 1 or 2, cyano, halogeno, and trifluoromethyl;
R9 and R10 are each independently selected from hydrogen and C1-4alkyl or R8 and R9 together with the nitrogen to which they are attached form a dihydroindolyl, or a dihydroquinolinyl group;
R11 is selected from carboxyl, tetrazolyl, alkyl sulphonyl carbamoyl, sulfo and sulfino;
Y is oxygen, sulphur or sulfonyl;
m is 0 or 1 and n is 0 or an integer from 1 to 4 with the proviso that m and n cannot both be 0 and when m is 1, n is 0;
or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof.
xe2x80x98C1-6alkylC1-4alkoxyxe2x80x99 typically means xe2x80x94(C1-6)alkylO(C1-4)alkyl, a preferred example of which is xe2x80x94CH2OCH3. xe2x80x98C1-6alkylaminoC1-6alkylxe2x80x99 typically means xe2x80x94(C1-6)alkylNH(C1-6)alkyl, a preferred example of which is xe2x80x94CH2NHC2H5.
xe2x80x98Arylxe2x80x99 typically means phenyl or naphthyl, preferably phenyl. xe2x80x98Heteroarylxe2x80x99 means an aromatic 5 or 6 membered ring with up to four ring heteroatoms selected from nitrogen, oxygen and sulphur. Examples of xe2x80x98heteroarylxe2x80x99 include pyrrolyl, fluranyl, thienyl, imidazolyl, thiadiazolyl, thiazolyl, isoxazolyl, pyridinyl, pyridyl and pyrimidimyl.
Bicyclic heteroaryl ring system means an aromatic 5,6-, 6,5 or 6,6 fused ring system wherein one or both rings contain ring heteroatoms. The ring system may contain up to three heteroatoms, independently selected from oxygen, nitrogen and sulphur. When the ring system contains more than one heteroatom at least one such heteroatom is nitrogen. An example of preferred bicyclic heteroaryl ring systems are isoquinolyl, benzothiazolyl or benzoimidazolyl.
9 or 10 membered bicyclic ring system means an aromatic 6 membered ring fused to a 5 or 6 membered ring, preferably a 5 or 6 membered saturated ring, optionally substituted with at least one heteroatom, preferably oxyygen, and linked to the nitrogen to which it is attached via a ring carbon on the aromatic 6 membered ring. A preferred example for R8 is tetrahydronaphthalyl. When R2, R3 and the phenyl to which they are attached form such a 9 or 10 membered bicyclic ring system, preferred groups are dihydrobenzofuranyl and dihydrobenzopyranyl.
R2 and R3 are preferably independently selected from hydrogen, C1-6 alkyl, C1-4alkoxy, trifluoromethyl and halogeno and, more preferably are independently selected from methyl, methoxy, isopropoxy, trifluoromethyl, fluoro, bromo and chloro.
A preferred group for R4 is C1-2alkyl. R5 is preferably selected from hydrogen, methyl and isopropyl. Preferably R6 is selected from C1-4 alkyl and C1-4alkylS(C1-4)alkyl and is, more preferably, selected from xe2x80x94CH2CH(CH3)(CH3) and xe2x80x94CH2CH2SCH3. R7 is preferably selected from C2-6 alkenyl, 1,3-benzodioxol-5-yl, and 1-isopropyl-2-methylpropyl acetyl and is, more preferably, selected from allyl and 1,3-benzodioxol-5-yl.
R8 is preferably phenyl, thienyl, pyridyl, thiadiazol, isoxazolyl, thiazolyl, 5,6,7,8-tetrahydronapthalyl, isoquinolyl, 1,3-benzoimidazolyl and 1,3-benzothiazolyl each optionally substituted with up to two substituents which are preferably and independently selected from C1-4alkyl, more preferably methyl, halogeno, more preferably fluoro, chloro and bromo; xe2x80x94COOH, xe2x80x94CH2OH, hydroxy and methylthio.
R9 and R10 are each independently preferably selected from hydrogen and methyl or R8 and R9 together with the nitrogen to which they are attached form 2,3-dihydro-1H-indol-1-yl, or 3,4-dihydro-1 (2H)-quinolinyl. R11 is preferably COOH. In a preferred embodiment n is 1 and m is 0. Y is preferably oxygen.
Preferred compounds according to the invention are those of formula (III) 
where R2 to R8, Y, m and n are as hereinbefore defined.
More preferred are those compounds where R2 is C1-4alkoxy, especially methoxy, R3, R5 and R10 are each independently hydrogen; R4 is C1-4 alkyl; R6 is selected from C1-4alkyl and C1-4alkylS(C1-4)alkyl and is especially xe2x80x94CH2CH(CH3)(CH3) or xe2x80x94CH2CH2SCH3; R7 is 1,3-benzodioxol-5-yl; R8 is aryl or heteroaryl each optionally substituted with one substitutent selected from C1-6alkyl, especially methyl, CH2OH, halogeno, especially chloro or fluoro, and hydroxy and R9 is hydrogen or C1-4alkyl or R8 and R9 together with the nitrogen to which they are attached form a dihydroindolyl or a dihydroquinolinyl; and m and n are 0 or 1 with the proviso that n and m cannot both be 0 or 1 and most preferably m is 0 and n is 1.
Particularly preferred compounds include 4-Nxe2x80x2-(2-methylphenyl)urea)-3-methoxy phenoxyacetyl (leucine-3-amino-(3,4-methylenedioxy)phenylpropionic acid)amide;
4-(Nxe2x80x2-phenylurea)-3-methoxy phenoxyacetyl (leucine-3-amino-(3,4-methylenedioxy)phenylpropionic acid)amide; 4-(Nxe2x80x2-(2-chlorophenyl)urea)-3-methoxy phenoxyacetyl (leucine-3-amino-(3,4-methylenedioxy)phenylpropionic acid)amide;
7-(Nxe2x80x2-(2-methylphenyl)urea)-2,3-dihydrobenzofuranyl-4-oxyacetyl(leucine-3-amino-(3,4-methylenedioxy)phenylpropionic acid)amide; 4-(Nxe2x80x2-(2-hydroxymethylphenyl)urea)-3-methoxy phenoxyacetyl (leucine-3-amino-(3,4-methylenedioxy)phenylpropionic acid)amide;
4-[(2,3-dihydro-1H-indol-1ylcarbonyl)amino]-3-methoxy phenoxyacetyl (leucine-3-amino-(3,4-methylenedioxy)phenylpropionic acid)amide; 4-(Nxe2x80x2-(2-fluorophenyl)urea)-3-methoxy phenoxyacetyl (leucine-3-amino-(3,4-methylenedioxy)phenylpropionic acid)amide;
4-(Nxe2x80x2-(2-hydroxy-6-methylphenyl)urea)-3-methoxy phenoxyacetyl (leucine-3-amino-(3,4-methylenedioxy)phenylpropionic acid)amide; and 4-(Nxe2x80x2-(2-methylphenyl)urea)-3-isopropoxy phenoxyacetyl (leucine-3-amino-(3,4-methylenedioxy)phenylpropionic acid)amide.
The compounds of formulae (II) and (III) possess chiral centres, at xe2x80x94CHR6, and at xe2x80x94CHR7. When R6 is either xe2x80x94CH2CH(CH3)(CH3) or xe2x80x94CH2CH2SCH3 and, therefore, the compounds of the invention of formulae (II) and (III) contains leucine or methionine as a sub-unit, the latter are in their proteinogenic (or natural) configuration. The present invention covers all diasteroisomers that inhibit the interaction between VCAM-1 and fibronectin with integrin VLA-4.
According to another aspect of the present invention there is provided a compound of formula (IV) 
wherein:
R1a is in the para or meta position and is 
xe2x80x83where
R2a and R3a are each independently selected from hydrogen, nitro, C1-6 alkyl, C3-6 cycloalkyl, C2-6alkenyl, C2-6alkynyl, C1-4 alkoxy, C1-6 alkylamino, C1-4alkoxylC1-6alkyl, C1-6alkylaminoC1-6alkyl, cyano, halogeno, trifluoromethyl, xe2x80x94CO2R7a and xe2x80x94CON R7a R8a where R7a and R8a are independently selected from hydrogen or C1-6 alkyl;
R4a is selected from C1-6 alkyl, C1-6alkoxy-substituted(C1-6)alkyl, and C1-6alkylS(C1-6)alkyl;
R5a is selected from C1-6alkyl, C2-6 alkenyl, 1,3-benzodioxol-5-yl and aryl optionally substituted by at least one substituent selected from C1-4 alkoxy, C1-6 alkyl, cyano, halogeno, and trifluoromethyl;
R6a is aryl or heteroaryl and the ring is optionally substituted with up to two substituents, which may be same or different, selected from C1-6 alkyl, C1-4 alkoxy, C1-4alkoxyC1-6alkyl, C1-6alkylaminoC1-6alkyl, cyano, halogeno, and trifluoromethyl;
Ya is oxygen or sulphur; and
na is an integer from 1 to 4;
or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof.
In compounds of formula (IV), xe2x80x98arylxe2x80x99 typically means phenyl or naphthyl, preferably phenyl. xe2x80x98Heteroarylxe2x80x99 means an aromatic 5 or 6 membered ring with up to four ring heteroatoms selected from nitrogen, oxygen and sulphur. Examples of xe2x80x98heteroarylxe2x80x99 include pyrrolyl, furanyl, thienyl, imidazolyl, thiazolyl, pyridinyl and pyrimidimyl.
R2a and R3a are preferably independently selected from hydrogen, C1-6 alkyl, trifluoromethyl and halogeno and, most preferably, are independently selected from methyl, trifluoromethyl and chloro. R4a is preferably selected from C1-4 alkyl and C1-4alkylS(C1-4)alkyl and is, more preferably, selected from xe2x80x94CH2CH(CH3)(CH3) and xe2x80x94CH2CH2SCH3. Preferably, R5a is selected from C2-6 alkenyl, more preferably allyl, and 1,3-benzodioxol-5-yl. R6a is preferably phenyl with up to two substituents which are preferably and independently selected from C1-4 alkyl, most preferably methyl, and halogeno, most preferably chloro and bromo. The preferred value for n is 1.
The compounds of formula (IV) of the present invention possess chiral centres, at xe2x80x94CHR4a, and at xe2x80x94CHR5a. When R4a is either xe2x80x94CH2CH(CH3)(CH3) or xe2x80x94CH2CH2SCH3 and, therefore, the compound of the invention of formula (IV) contains leucine or methionine as a sub-unit, the latter are in their proteinogenic (or natural) configuration. The present invention covers all diasteroisomers that inhibit the interaction between VCAM-1 and fibronectin with integrin VLA-4.
Pharmaceutically acceptable salts of the compounds of formulae (II), (III) and (IV) include acid addition salts such as salts formed with mineral acids, for example, hydrogen halides such as hydrogen chloride and hydrogen bromide, sulphonic and phosphonic acids; and salts formed with organic acids, especially citric, maleate, acetic, oxalic, tartaric, mandelic, p-toluenesulphonic, methanesulphonic acids and the like. In another aspect, suitable salts are base salts such as alkali metals salts, for example, sodium and potassium; alkaline earth metal salts such as magnesium and calcium; aluminium and ammonium salts; and salts with organic bases such as ethanolamine, methylamine, diethylamine, isopropylamine, trimethylamine and the like. Such salts may be prepared by any suitable method known in the art.
In vivo hydrolysable esters are those pharmaceutically acceptable esters that hydrolyse in the human body to produce the parent compound. Such esters can be identified by administering, for example intravenously to the test animal, the compound under test and subsequently examining the test animal""s body fluids. Suitable in vivo hydrolysable esters for hydroxy include acetyl and for carboxyl include, for example, C1-6alkoxy methyl esters for example methoxymethyl, C1-6alkanoyloxymethyl esters for example pivaloyloxymethyl, phthalidyl esters, C3-8 cycloalkoxycarbonyloxyC1-6alkyl esters for example 1-cyclohexylcarbonyloxyethyl; 1,3-dioxolan-2-ylmethyl esters for example 5-methyl-1,3-dioxolan-2-ylmethyl; and C1-6alkoxycarbonyloxyethyl esters for example 1-methoxycarbonyloxyethyl.
The activities of the compounds of this invention to inhibit the interaction between VCAM-1 and fibronectin with integrin VLA-4 may be determined using a number of in vitro and in vivo screens. They show improved potency compared to prior art compounds.
For example, the compounds according to the invention preferably have an IC50 of  less than 10 xcexcnM, more preferably  less than 1 xcexcM in the MOLT-4 cell/Fibronectin assay hereinafter described.
Preferred compounds have shown activity in a number of in vivo screens in mice, for example, delayed-type hypersensitivity (DTH) responses induced by ovalbumin in the footpad and collagen-induced arthritis.
In order for it to be used, a compound of formulae (II), (III) or (IV) or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof is typically formulated as a pharmaceutical composition in accordance with standard pharmaceutical practice.
Thus, according to a further aspect of the invention there is provided a pharmaceutical composition which comprises a compound of formula (II), (III) or (IV) or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof and a pharmaceutically acceptable carrier.
The pharmaceutical composition of this invention may be in a form suitable for oral use, for example a tablet, capsule, aqueous or oily solution, suspension or emulsion; for nasal use, for example a snuff, nasal spray or nasal drops; for vaginal or rectal use, for example a suppository; for administration by inhalation, for example as a finely divided powder or a liquid aerosol; for sub-lingual or buccal use, for example a tablet or capsule; or for parenteral use (including intravenous, subcutaneous, intramuscular, intravascular or infusion), for example a sterile aqueous or oily solution or suspension, or a depot formulation with drug incorporated in a biodegradable polymer. The composition may be in a form suitable for topical administration such as for example creams, ointments and gels. Skin patches are also contemplated. For these purposes, the composition of this invention may be formulated by means known in the art, such as for example, as described in general terms, in Chapter 25.2 of Comprehensive Medicinal Chemistry, Volume 5, Editor Hansch et al, Pergamon Press 1990.
Furthermore, the pharmaceutical composition of the present invention may contain one or more additional pharmacological agents suitable for treating one or more disease conditions referred to hereinabove in addition to the compounds of the present invention. In a further aspect, the additional pharmacological agent or agents may be co-administered, either simultaneously or sequentially, with the pharmaceutical composition of the invention.
The composition of the invention will normally be administered to humans such that the daily dose will be 0.01 to 75 mg/kg body weight and preferably 0.1 to 15 mg/kg body weight. A preferred composition of the invention is one suitable for oral administration in unit dosage form for example a tablet or capsule which contains from 1 to 1000 mg and preferably 10 to 500 mg of a compound according to the present invention in each unit dose.
Thus, according to yet another aspect of the invention, there is provided a compound of formulae (II), (III) or (IV) or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof for use in a method of therapeutic treatment of the human or animal body.
In yet a further aspect of the invention the present invention provides a method of treating a disease mediated by the interaction between VCAM-1 and/or fibronectin and the integrin receptor VLA-4 in need of such treatment which comprises administering to said warm-blooded mammals an effective amount of a compound of formulae (II), (III) or (IV) or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof.
The present invention also provides the use of a compound of formulae (II), (III) or (IV) or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof in the production of a medicament for use in the treatment of a disease or medical condition mediated by the interaction between fibronectin and/or VCAM-1 (especially VCAM-1) and the integrin receptor VLA-4.
In one embodiment of the invention the manumal in need of treatment is suffering from multiple sclerosis, rheumatoid arthritis, asthma, coronary artery disease or psoriasis.
In another aspect of the invention, there is provided a process for preparing a compound of formula (II) where R11 is COOH or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof which process comprises coupling together
i) a compound of formula (V) 
xe2x80x83and a compound of formula (VI)
NHR5xe2x80x94CHR6xe2x80x94CONHxe2x80x94CHR7xe2x80x94CH2xe2x80x94COOHxe2x80x83xe2x80x83(VI)
xe2x80x83or
ii) a compound of formula (VII) 
xe2x80x83and a compound of formula (VIII)
NH2xe2x80x94CHR7xe2x80x94CH2xe2x80x94COOHxe2x80x83xe2x80x83(VIII)
xe2x80x83wherein L and L1 are leaving groups and any functional group is optionally protected; and thereafter, if necessary:
a) removing any protecting group; and
b) forming a pharmaceutically acceptable salt or in-vivo hydrolysable ester.
The reactions of (V) and (VI) or (VII) and (VIII) are performed under standard coupling conditions for forming peptide bonds. They can be performed either on a solid support (Solid Phase Peptide Synthesis) or in solution using normal techniques used in the synthesis of organic compounds. With the exception of the solid support, all the other protecting groups, coupling agents, deblocking reagents and purification techniques are similar in both the solid phase and solution phase peptide synthesis techniques.
During the reaction, amino acid functional groups may, if necessary, be protected by protecting groups, for example Boc. Such groups can be cleaved when necessary using standard techniques such as acid or base treatment.
Suitable protecting groups for the protection of the carboxyl groups include esters.
Coupling reagents for forming peptide bonds include the commonly used azide, symmetrical anhydride, mixed anhydride and various active esters and carbodiimides. In the case of carbodiimides, additives such as 1-hydroxybenzotriazole and N-hydroxysuccinimide may also be added. Other coupling reagents include 1H-benzotriazole-1-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate (PyBOP), (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)] and O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU).
The coupling reactions can be performed at temperatures between xe2x88x9220xc2x0 C. to 40xc2x0 C. The time of the reaction can vary such as between 10 minutes and 24 hours. Suitable purification methods for the intermediates and final products include chromatographic techniques such as high pressure liquid chromatography (HPLC) along with many other standard techniques used in organic chemistry (e.g. solvent extraction and crystallisation).
The following abbreviations are used:
Preferred coupling conditions for reacting compounds of formula (V) and (VI) or (VII) and (VIII) are, in particular,
a) HATU/DIPEA/DMF
b) HOBT/WSCDI/DIPEA/DMF
c) HOBT/WSCDI/DIPEA/N-methylmorpholine
Compounds of formula (VI) may be prepared by reacting a compound of formula (IX)
NHR5CHR6COLxe2x80x2xe2x80x83xe2x80x83(IX)
with a compound of formula (VIII). Preferably the compound of formula (IX) is in the form of Boc-amino acid or Boc-amino acid-OSu and the coupling reagents are selected from
d) HATU/DMF/DIPEA;
e) HOBT/WSCDI/DIPEA/DMF/CH2Cl2; and
f) Et3N/CH2Cl2;
The protecting group may be removed using any suitable reagent known in the art, a particularly preferred example of which is trifluoroacetic acid
Compounds of formula (VI) may be prepared by reacting a compound of formula (V) and a compound of formula (IX) in a standard manner.
Exemplary methods of preparing compounds of formula (VIII) are as follows. When R7 is aryl or 1,3-benzodioxol-5-yl, the following method may used. 
where R15 is a substituent selected from C1-4 alkoxyl, C1-6 alkyl, cyano, halogeno and trifluoromethyl; or 
forms 1,3-benzodioxol-5-yl and p is an integer from 1 to 4.
When R7 is C2-6 alkenyl the following method may be used 
As will be appreciated from the art BF3Et2O can be replaced by other known Lewis acids, 
replaced by, for example, by allyl bromide, and HCl/MeOH may be replaced by, for example, HBr/acetic acid.
An exemplary method of preparing a compound of formula (V) where Y is oxygen, m is 0, n is 1 and R8 is a 6 membered aromatic ring is as follows, where R16 and R17 are independently selected from hydrogen, C1-6 alkyl, C1-4 alkoxy, C1-4alkylthio, C1-6alkylC1-4alkoxy, C1-6alkylaminoC1-6alkyl, hydroxy, xe2x80x94CO2H, xe2x80x94(CH2)pOH where p is 1 or 2, cyano, halogeno and trifluoromethyl: 
In the first step of the reaction bases other than sodium methoxide may be used and esters other t-butyl esters could be used but these will typically require a final basic rather than acid hydrolysis to form the final product. For compounds of formula (V) where m is 0 and n is 2 the phenoxide ion (XVII) will be added to an acrylic ester. A further route for preparing compounds of formula (XXI) involves reacting a compound of formula (XIX) with triphosgene and then an amine of formula (XXII). Alternatively the triphosgene can be reacted with amine (XXII) and then the compound of formula (XIX). 
Compounds of formula (XXI) may also be prepared according to the following reaction: 
When Y is sulphur an exemplary method of preparing the compounds of invention is as follows. A compound of formula (V) can be formed by reacting phenyl isocyanate, optionally substituted on the phenyl ring, with a 2-(4-aminophenylthio)acetic acid. To this is added a coupling reagent and a compound of formula (VI) and (VII).
When Y is sulphonyl an exemplary method of preparing the compounds of the invention is as follows. A compound of formula (II) in which Yxe2x95x90S is oxidised by treatment with Oxone, m-chloroperbenzoic or other suitable oxidant. In the process the intermediate sulphoxide (Yxe2x95x90SO) may be isolated initially and further more vigorous conditions employed to give the sulphonyl derivatives.
When R11 is acyl sulphonamide (xe2x80x94CONHSO2Rx) in a compound of formula (II) an exemplary method of preparing these compounds is as follows. A compound of formula (II) in which R11 is xe2x80x94CO2H is treated with a sulphonamide of formula RxSO2NH2 in the presence of 4-dimethylaminopyridine and a carbodiimide.
Compounds of the invention may contain more than two units which are to be joined together by the formation of amide links. Where such units are present, the person skilled in the art will be aware of the preferred order of joining such units together.
The invention is further illustrated by the following biological test methods, data and non-limiting Examples.
Table 1 refers to examples 13 to 76. It gives the structure of the final materials and their analysis. It also refers to the method by which they were prepared by reference to a code.